The laboratory diagnosis of infection requires the demonstration—either direct or indirect—of viral, bacterial, fungal, or parasitic agents in tissues, fluids, or excreta of the host. Clinical microbiology laboratories are responsible for processing these specimens and also for determining the antibiotic susceptibility of bacterial and fungal pathogens. Traditionally, detection of pathogenic agents has relied largely on either the microscopic visualization of pathogens in clinical material or the growth of microorganisms in the laboratory. Identification generally is based on phenotypic characteristics such as fermentation profiles for bacteria, cytopathic effects in tissue culture for viral agents, and microscopic morphology for fungi and parasites. These techniques are reliable but are often time-consuming. Increasingly, the use of nucleic acid probes is becoming a standard method for detection, quantitation, and/or identification in the clinical microbiology laboratory, gradually replacing phenotypic characterization and microscopic visualization methods.
Reappraisal of the methods employed in the clinical microbiology laboratory has led to the development of strategies for detection of pathogenic agents through nonvisual biologic signal detection systems. Much of this methodology is based on the use of either electronic detection systems involving relatively inexpensive but sophisticated computers or nucleic acid probes directed at specific DNA or RNA targets. This chapter discusses both the methods that are currently available and those that are being developed.
A biologic signal is a material that can be reproducibly differentiated from other substances present in the same physical environment. Key issues in the use of a biologic (or electronic) signal are distinguishing it from background noise and translating it into meaningful information. Examples of biologic signals applicable to clinical microbiology include structural components of bacteria, fungi, and viruses; specific antigens; metabolic end products; unique DNA or RNA base sequences; enzymes; toxins or other proteins; and surface polysaccharides.
A detector is used to sense a signal and discriminate between that signal and background noise. Detection systems range from the trained eyes of a technologist assessing morphologic variations to sensitive electronic instruments such as gas-liquid chromatographs coupled to computer systems for signal analysis. The sensitivity with which signals can be detected varies widely. It is essential to use a detection system that discerns small amounts of signal even when biologic background noise is present—i.e., that is both sensitive and specific. Common detection systems include immunofluorescence; chemiluminescence for DNA/RNA probes; flame ionization detection of short- or long-chain fatty acids; and detection of substrate utilization or end-product formation as color changes, of enzyme activity as a change in light absorbance, of turbidity changes as a measure of growth, of cytopathic effects in cell lines, and of particle agglutination as a measure of antigen presence.
Amplification enhances the sensitivity with which weak signals can be detected. The most common microbiologic amplification technique is growth of a single bacterium into a discrete colony on an agar plate or into a suspension containing many identical organisms. The advantage of growth as an amplification method is that it requires only an appropriate growth medium; the disadvantage is the amount of time required. More rapid specific amplification of biologic signals can be achieved with techniques such as polymerase chain reaction (PCR), ligase chain reaction (LCR), and transcription-mediated amplification (TMA), all of which target the pathogen's DNA/RNA; enzyme immunoassays (EIAs, for antigens and antibodies); electronic amplification (for gas-liquid chromatography assays); antibody capture methods (for concentration and/or separation); and selective filtration or centrifugation. Although a variety of methods are available for the amplification and detection of biologic signals in research, thorough testing is required before these methods are validated as diagnostic assays.
The field of microbiology has been defined largely by the development and use of the microscope. The examination of specimens by microscopic methods rapidly provides useful diagnostic information. Staining techniques permit organisms to be seen more clearly.
The simplest method for microscopic evaluation is the wet mount, which is used, for example, to examine cerebrospinal fluid (CSF) for the presence of Cryptococcus neoformans, with India ink as a background against which to visualize large-capsuled yeast cells. Wet mounts with dark-field illumination also are used to detect spirochetes from genital lesions and to reveal Borrelia or Leptospira in blood. Skin scrapings and hair samples can be examined with the use of either 10% KOH wet-mount preparations or the Calcofluor white method and ultraviolet illumination to detect fungal elements as fluorescing structures. Staining of wet mounts—e.g., with lactophenol cotton blue stain for fungal elements—often is used for morphologic identification. These techniques enhance signal detection and decrease the background, making it easier to identify specific fungal structures.
Without staining, bacteria are difficult to see at the magnifications (400× to 1000×) used for their detection. Although simple one-step stains can be used, differential stains are more common. Gram's stain differentiates between organisms with thick peptidoglycan cell walls (gram-positive) and those with thin peptidoglycan cell walls and outer membranes that can be dissolved with alcohol or acetone (gram-negative). Cellular morphology and Gram's stain characteristics often can be used to categorize stained organisms into groups such as streptococci, staphylococci, and clostridia (Fig. e22-1).
Interpretation of Gram's stain.
Gram's stain is particularly useful for examining sputum for polymorphonuclear leukocytes (PMNs) and bacteria. Sputum specimens from immunocompetent patients with ≥25 PMNs and <10 epithelial cells per low-power field often provide clinically useful information. However, the presence in "sputum" samples of >10 epithelial cells per low-power field and of multiple bacterial types suggests contamination with oral microflora. Despite the difficulty of discriminating between normal microflora and pathogens, Gram's stain may prove useful for specimens from areas with a large resident microflora if a useful ...